Polynucleotides and methods for improving plants

ABSTRACT

The invention provides methods and compositions for producing plant with altered biomass, the methods comprising the step of altering the expression and/or activity of the polypeptide comprising the sequence of SEQ ID NO:1, or a variant thereof, in a plant cell or plant. The invention also provides a polypeptide comprising the sequence of SEQ ID NO:1, and fragments of variants thereof the sequence. The invention also provides polynucleotides encoding such polypetide sequences. The invention also provides constructs, cells and plants comprising such polynucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority as a continuation of U.S. applicationSer. No. 13/528,558, filed Jun. 20, 2012, which itself is a continuationof U.S. application Ser. No. 12/324,664, filed Nov. 26, 2008, and claimspriority to U.S. Provisional Application No. 60/990,590, filed Nov. 27,2007. The priority applications are incorporated herein by reference intheir entirety.

REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSequence-Listing-JAMES161002C2.txt, created May 7, 2015, which is 76.8Kb in size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions and methods for producingplants with increased biomass.

2. Description of the Related Art

As the population of the world increases, a major goal of agriculturalresearch is to improve the biomass yield of crop and forage plantspecies.

Such improvements have until recently depended on selective breeding ofplants for desirable characteristics. However for many plants theheterogeneous genetic complements produced in off-spring do not resultin the same desirable traits as those of their parents, thus limitingthe effectiveness of selective breeding approaches.

Advances in molecular biology now make it possible to geneticallymanipulate the germplasm of both plants and animals. Genetic engineeringof plants involves the isolation and manipulation of genetic materialand the subsequent introduction of such material into a plant. Thistechnology has led to the development of plants that are capable ofexpressing pharmaceuticals and other chemicals, plants with increasedpest resistance, increased stress tolerance, and plants that expressother beneficial traits.

Whilst it is known in the art that certain growth factors may be appliedto increase plant size, the application of such growth factors is bothcostly and time consuming Thus, there exists a need for plants withincreased biomass relative to their cultivated counterparts.

It is an object of the invention to provide improved compositions and/ormethods for developing plant varieties with altered biomass or at leastto provide the public with a useful choice.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a method for producing a plantwith altered biomass, the method comprising transformation of a plantwith a:

-   -   a) a polynucleotide including a sequence encoding of a        polypeptide with the amino acid sequence of SEQ ID NO:1 or a        variant of the polypeptide; or    -   b) a polynucleotide comprising a fragment, of at least 15        nucleotides in length, of the polynucleotide of a); or    -   c) a polynucleotide comprising a compliment, of at least 15        nucleotides in length, of the polynucleotide of a); or d) a        polynucleotide comprising a sequence, of at least 15 nucleotides        in length, capable of hybridising to the polynucleotide of a)        under stringent conditions.

Preferably the polynucleotide is included as part of a geneticconstruct.

In one embodiment the variant has at least 70% sequence identity to apolypeptide with the amino acid sequence of SEQ ID NO: 1.

In a further embodiment the variant comprises the amino acid sequence ofSEQ ID NO: 20.

In a further embodiment the variant is derived from a plant species andcomprises the amino acid sequence of SEQ ID NO: 20.

In a further embodiment the variant is derived from a dicotyledonousplant species and comprises the amino acid sequence of SEQ ID NO: 21.

Preferably the variant is capable of modulating biomass in a plant

In a further embodiment the polynucleotide of a) encodes a polypeptidewith the amino acid sequence of SEQ ID NO: 1.

Preferably expression of the polynucleotide in the plant results indown-regulation of an endogenous polynucleotide/polypeptide capable ofmodulating biomass production in the plant.

Preferably the reduced expression is effected by antisense suppression,sense suppression or RNA interference.

Preferably the plant produced has increased biomass relative to asuitable control plant.

In a further aspect the invention provides a method for producing aplant with increased biomass, the method comprising transformation of aplant with a polynucleotide with sufficient sequence similarity to anendogenous nucleic acid encoding a polypeptide with the sequence of SEQID NO:1 or a variant thereof, such that expression of the polynucleotideresults in inhibition of expression of the endogenous nucleic acid.

Preferably the polynucleotide is included as part of a geneticconstruct.

In one embodiment the variant has at least 70% sequence identity to apolypeptide with the amino acid sequence of SEQ ID NO: 1.

In a further embodiment the variant comprises the amino acid sequence ofSEQ ID NO: 20.

In a further embodiment the variant is derived from a plant species andcomprises the amino acid sequence of SEQ ID NO: 20.

In a further embodiment the variant is derived from a dicotyledonousplant species and comprises the amino acid sequence of SEQ ID NO: 21.

Preferably the variant is capable of modulating biomass in a plant.

In a further embodiment the polypeptide has the sequence of SEQ ID NO:1

In a further aspect the invention provides a method of producing a plantwith altered biomass, the method comprising transformation of a plantcell or plant with a:

-   -   a) a polynucleotide comprising the nucleotide sequence of SEQ ID        NO:10, or a variant thereof; or    -   b) a polynucleotide comprising a fragment, of at least 15        nucleotides in length, of the polynucleotide of a); or    -   c) a polynucleotide comprising a complement, of at least 15        nucleotides in length, of the polynucleotide of a); or    -   d) a polynucleotide comprising a sequence, of at least 15        nucleotides in length, capable of hybridising to the        polynucleotide of a) under stringent conditions.

Preferably the polynucleotide is included as part of a geneticconstruct.

Preferably the variant encodes a polypeptide capable of modulatingbiomass in a plant

In one embodiment the polynucleotide of a) comprises the sequence of SEQID NO:10. Preferably expression of the polynucleotide in the plantresults in down-regulation of an endogenous polynucleotide/polypeptidecapable of modulating biomass production in the plant.

Preferably the down-regulation is effected by antisense suppression,sense suppression or RNA interference.

Preferably the plant produced by the method of the invention hasincreased biomass relative to a suitable control plant.

In a further aspect the invention provides a method for producing aplant with increased biomass the method comprising transformation of aplant with a polynucleotide with sufficient sequence similarity to anendogenous nucleic acid with the sequence of SEQ ID NO:10 or a variantthereof, such that in expression of the polynucleotide results ininhibition of expression of the endogenous nucleic acid.

Preferably the polynucleotide is included as part of a geneticconstruct.

In one embodiment the variant has at least 70% sequence identity withthe full-length coding sequence of SEQ ID NO: 10.

In a further embodiment the variant encodes a polypeptide comprising theamino acid sequence of SEQ ID NO: 20.

In a further embodiment the variant is derived from a plant species andencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:20.

In a further embodiment the variant is derived from a dicotyledonousplant species and encodes a polypeptide comprising the amino acidsequence of SEQ ID NO: 21.

Preferably the variant encodes a polypeptide capable of modulatingbiomass in a plant

In a further embodiment the endogenous nucleic acid comprises thefull-length coding sequence of SEQ ID NO:10.

In a further aspect the invention provides a method for producing aplant cell or plant with altered biomass, the method comprising reducingthe expression or activity of a polypeptide including the amino acidsequence of SEQ ID NO: 1 or variant thereof.

In one embodiment the variant has at least 70% sequence identity to apolypeptide with the amino acid sequence of SEQ ID NO: 1.

In a further embodiment the variant comprises the amino acid sequence ofSEQ ID NO: 20.

In a further embodiment the variant is derived from a plant species andcomprises the amino acid sequence of SEQ ID NO: 20.

In a further embodiment the variant is derived from a dicotyledonousplant species and comprises the amino acid sequence of SEQ ID NO: 21.

In a further embodiment the polypeptide has the sequence of SEQ ID NO:1

In a further aspect the invention provides a method of producing a plantwith altered biomass the method comprising the step of reducing theexpression or activity in a plant cell or plant of a polypeptidecomprising the sequence of SEQ ID NO: 20.

In one embodiment the polypeptide comprises the sequence of SEQ ID NO:21.

In a further embodiment the the polypeptide comprises the sequence ofwith at least 70% identity to the sequence of SEQ ID NO: 1.

In a further embodiment the polypeptide comprises the sequence of SEQ IDNO: 1.

In a further embodiment the a polynucleotide capable of hybridisingunder stringent conditions to an endogenous nucleic acid encoding thepolypeptide is introduced into the plant cell or plant to effect reducedexpression of the polypeptide.

In a further embodiment the endogenous nucleic acid comprises a sequencewith at least 70% identity to the full-length coding sequence of SEQ IDNO: 10.

In a further embodiment the endogenous nucleic acid comprises thefull-length coding sequence of SEQ ID NO: 10.

In a further embodiment the polynucleotide comprises at least 15contiguous nucleotides of a sequence with at least 70% identity to thesequence of SEQ ID NO: 10.

In a further embodiment the polynucleotide comprises at least 15contiguous nucleotides of SEQ ID NO: 10.

In a further aspect the invention provides a plant cell or plantproduced by a method of the invention.

Preferably the plant produced by the method of the invention hasincreased biomass production relative to a suitable control plant.

Preferably the plant produced by the method of the invention has anincreased number of tillers relative to a suitable control plant.

In a further aspect the invention provides an isolated polynucleotidehaving at least 71% sequence identity to a nucleotide sequence thatencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:1.

Preferably the polynucleotide encodes a polypeptide capable ofmodulating biomass in a plant

In one embodiment the polypeptide comprises the amino acid sequence ofSEQ ID NO:1

In a further embodiment the nucleotide sequence comprises the sequenceof SEQ ID NO:10.

In a further embodiment said nucleotide sequence comprises thefull-length coding sequence of SEQ ID NO:10.

In a further aspect the invention provides an isolated polynucleotidethat encodes a polypeptide comprising an amino acid sequence SEQ ID NO:1.

In one embodiment the polynucleotide comprises the sequence of SEQ IDNO:10.

In a further embodiment the polynucleotide comprises the full-lengthcoding sequence of SEQ ID NO:10.

In a further aspect the invention provides an isolated polynucleotidecomprising the full-length coding sequence of SEQ ID NO: 10 or a variantthereof, wherein the variant is derived from ryegrass or fescue, andencodes a polypeptide capable of modulating biomass in a plant.

In one embodiment the variant has at least 70% sequence identity to thefull-length coding sequence of SEQ ID NO:10.

In one embodiment the isolated polynucleotide comprises the sequence ofSEQ ID NO:10.

In a further aspect the invention provides an isolated polypeptidehaving at least 90% sequence identity to the amino acid sequence of SEQID NO: 1, wherein the polypeptide is capable of modulating biomass in aplant.

In one embodiment the isolated polypeptide the amino acid sequence ofSEQ ID NO: 1.

In a further aspect the invention provides an isolated polynucleotideencoding a polypeptide of the invention.

In a further aspect the invention provides an isolated polynucleotidecomprising:

-   -   a) a polynucleotide comprising a fragment, of at least 15        nucleotides in length, of a polynucleotide of the invention; or    -   b) a polynucleotide comprising a complement, of at least 15        nucleotides in length, of the polynucleotide of the invention;        or    -   c) a polynucleotide comprising a sequence, of at least 15        nucleotides in length, capable of hybridising to the        polynucleotide of the invention.

In a further aspect the invention provides a genetic construct whichcomprises a polynucleotide of the invention.

In one embodiment the genetic construct is an expression construct.

In a further aspect the invention provides a vector comprising anexpression construct or genetic construct of the invention.

In a further aspect the invention provides a host cell geneticallymodified to express a polynucleotide of the invention, or a polypeptideof the invention.

In a further aspect the invention provides a host cell comprising anexpression construct or genetic construct of the invention.

In a further aspect the invention provides a plant cell geneticallymodified to express a polynucleotide of the invention, or a polypeptideof the invention.

In a further aspect the invention provides a plant cell which comprisesan expression construct of the invention or the genetic construct of theinvention.

Preferably the expression construct is capable of expressing thepolynucleotide, resulting in inhibition of expression of an endogenouspolynucleotide/polypeptide which is capable of modulating biomassproduction in the plant.

In a further aspect the invention provides a plant which comprises aplant cell of the invention.

In a further aspect the invention provides a method for selecting aplant with altered biomass, the method comprising testing of a plant foraltered expression of a polynucleotide of the invention.

In a further aspect the invention provides a method for selecting aplant with altered biomass, the method comprising testing of a plant foraltered expression of a polypeptide of the invention.

In a further aspect the invention provides a plant cell or plantproduced by the method of the invention.

In a further aspect the invention provides a plant selected by themethod of the invention.

In a further aspect the invention provides an antibody raised against apolypeptide of the invention.

The polynucleotides and polynucleotide variants, of the invention may bederived from any species and/or may be produced recombinantly orsynthetically.

In one embodiment the polynucleotide or variant, is derived from a plantspecies.

In a further embodiment the polynucleotide or variant, is derived from agymnosperm plant species.

In a further embodiment the polynucleotide or variant, is derived froman angiosperm plant species.

In a further embodiment the polynucleotide or variant, is derived from afrom dicotyledonous plant species.

In a further embodiment the polynucleotide or variant, is derived from amonocotyledonous plant species.

The polypeptide and polypeptide variants, of the invention may bederived from any species and/or may be produced recombinantly orsynthetically.

In one embodiment the polypeptide or variant, is derived from a plantspecies.

In a further embodiment the polypeptide or variant, is derived from agymnosperm plant species.

In a further embodiment the polypeptide or variant, is derived from anangiosperm plant species.

In a further embodiment the polypeptide or variant, is derived from afrom dicotyledonous plant species.

In a further embodiment the polypeptide or variant, is derived from amonocotyledonous plant species.

The plant cell or plant may be derived from any plant species.

In a further embodiment the plant cell or plant, is derived from agymnosperm plant species.

In a further embodiment the plant cell or plant, is derived from anangiosperm plant species.

In a further embodiment the plant cell or plant, is derived from a fromdicotyledonous plant species.

In a further embodiment the plant cell or plant, is derived from amonocotyledonous plant species.

Preferred dicotyledonous genera include: Amygdalus, Anacardium, Arachis,Brassica, Cajanus, Cannabis, Carthamus, Carya, Ceiba, Cicer, Cocos,Coriandrum, Coronilla, Cossypium, Crotalaria, Dolichos, Elaeis, lycine,Gossypium, Helianthus, lathyrus, Lens, lespedeza, Linum Lotus, Lupinus,Macadamia, Medicago, Melilotus, Mucana, Olea, Onobrychis, Ornithopus,papaver, Phaseolus, Phoenix, Pistacia, Pitum, Prunus, Pueraria, ribes,Richinus, Sesamum, Theobroma, Trifolium, Trigonella, Vicia and Vigna.

Preferred dicotyledonous species include: Amygdalus communis, Anacardiumoccidentale, Arachis hypogaea, Arachis hypogea, Brassica napus Rape,Brassica, nigra, Brassica campestris, Cajanus cajan, Cajanus indicus,cannabis sativa, Carthamus tinctorius, Carya illinoinensis, Ceibapentandra, Cicer arietinum, Cocos nucifera, Coriandrum sativum,Coronilla varia, Cossypium hirsutum, Crotalaria juncea, Dolichos lablab,Elaeis guineensis, Gossypium arboreum, Gossypium nanking, Gossypiumbarbadense, Gossypium herbaceum, Gossypium, hirsutum, Glycine max,Glycine ussuriensis, Glycine gracilis, Helianthus annus, Lupinusangustifolius, Lupinus luteus, Lupinus matabilis, Lespedeza sericea,Lespedeza striate, Lotus uliginosus, Luthyrus sativus, Lens culinaris,Lespedeza stipulacea, Linum usitatissimum, Lotus corniculatus, Lupinusalbus, Medicago arborea, Medicago falcate, Medicago hispida, Medicagoofficinalis, medicago, sativa Alfalfa, medicago tribuloides, Macadamiaintegrifoniia, Medicago arabica, Melilotus albus, Mucuna prim:ens, Oleaeuropaea, Onobrychis viciifolia, Ornithopus sativus, Phaseolus aureus,Prunus cerasifera, Prunus cerasus, Phaseolus coccineus, Prunusdomestica, Phaseolus lumatus, Prunus maheleb, Phaseolus mango, Prunuspersica, Prunus pseudocerasus, Phaseolus vulgaris, Papaver soinniferum,Phaseolus acutifolius, Phoenix dactylifera, Pistacia vera, Pisumsativum, Prunus amygdalus, Prunus armeniaca, Pueraria thunbergiana,Ribes nigrum, Ribes rubrum, Ribes grossularia, Ricinus communis, Sesamumindicum, Trifolium augustifolium, Trifolium diffusum, Trifoliumhybridum, Trifolium incernatum, Trifolium ingrescens, Trifoliumpratense, Trifolium repens, Trifolium resupinatum, Trioliumsubterraneum, Theobroma cacao, Trifolium alexandrinum, Trigonellafoenumgraecum, Vicia angestifolia, Vicia atropurpurea, Vicia calcarata,Vicia dasycarpa, Vicia ervilia, Vaccinium oxycoccos, Vicia pannonica,Vigna sesquipedalis, Vigna sinensis, Vicia vollosa, Vicia faba, Viciasative and Vigna angularis.

Preferred monocotyledonous genera include: Agropyron, Allium,Alopecurus, Andropogon, Arrhenatherum, Asparagus, Avena, Bambusa,Bothrichloa, Bouteloua, Bromus, Calamovilfa, Cenchrus, Chloris,Cymbopogon, Cynodon, Dactylis, Dichanthium, Digitaria, Eleusine,Eragrostis, Fagopyrum, Festuca, Helianthus, Hordeum, Lolium, Miscanthis,Miscanthus x giganteus, Oryza, Panicum, Paspalum, Pennisetum, Phalaris,Phleum, Poa, Saccharum, Secale, Setaria, Sorgahastum, Sorghum, Triticum,Vanilla, X Triticosecale Triticale and Zea.

Preferred monocotyledonous species include: Agropyron cristatum,Agropyron desertorum, Agropyron elongatum, Agropyron intermedium,Agropyron smithii, Agropyron spicatum, Agropyron trachycaulum, Agropyrontrichophorum, Album ascalonicum, Album cepa, Album chinense, Alliumporrum, Album schoenoprasum, Album fistulosum, Album sativum, Alopecuruspratensis, Andropogon gerardi, Andropogon Gerardii, Andropogonscoparious, Arrhenatherum elatius, Asparagus officinalis, Avena nuda,Avena sativa, Bambusa vulgaris, Bothrichloa barbinodis, Bothrichloaischaemum, Bothrichloa saccharoides, Bouteloua curipendula, Boutelouaeriopoda, Bouteloua gracilis, Bromus erectus, Bromus inermis, Bromusriparius, Calamovilfa longifilia, Cenchrus ciliaris, Chloris gayana,Cymbopogon nardus, Cynodon dactylon, Dactylis glomerata, Dichanthiumannulatum, Dichanthium aristatum, Dichanthium sericeum, Digitariadecumbens, Digitaria smutsii, Eleusine coracan, Elymus angustus, Elymusjunceus, Eragrostis curvula, Eragrostis tef Fagopyrum esculentum,Fagopyrum tataricum, Festuca arundinacea, Festuca ovina, Festucapratensis, Festuca rubra, Helianthus annuus sunflower, Hordeumdistichum, Hordeum vulgare, Lolium multiflorum, Lolium perenn,Miscanthis sinensis, Miscanthus x giganteus, Oryza sativa, Panicumitalicium, Panicum maximum, Panicum miliaceum, Panicum purpurascens,Panicum virgatum, Panicum virgatum, Paspalum dilatatum, Paspalumnotatum, Pennisetum clandestinum, Pennisetum glaucum, Pennisetumpurpureum, Pennisetum spicatum, Phalaris arundinacea, Phleum bertolinii,Phleum pratense, Poa fendleriana, Poa pratensis, Poa. nemoralis,Saccharum officinarum, Saccharum robustum, Saccharum sinense, Saccharumspontaneum, Secale cereale, Setaria sphacelata, Sorgahastum nutans,Sorghastrum nutans, Sorghum dochna, Sorghum halepense, Sorghumsudanense, Sorghum vulgare, Sorghum vulgare, Triticum aestivum, Triticumdicoccum, Triticum durum, Triticum monococcum, Vanilla fragrans, XTriticosecale and Zea mays.

Preferred plants are forage plant species from a group comprising butnot limited to the following genera: Lolium, Festuca, Dactylis,Bromus,Trifolium, Medicago, Phleum, Phalaris, Holcus, Lotus, Plantago andCichorium.

Particularly preferred plants are from the genera Lolium and Trifolium.Particularly preferred species are Lolium perenne and Trifolium repens.

Particularly preferred monocotyledonous plant species are: Loliumperenne and Oryza sativa.

The term “plant” is intended to include a whole plant, any part of aplant, propagules and progeny of a plant.

The term ‘propagule’ means any part of a plant that may be used inreproduction or propagation, either sexual or asexual, including seedsand cuttings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Polynucleotides andFragments

The term “polynucleotide(s),” as used herein, means a single ordouble-stranded deoxyribonucleotide or ribonucleotide polymer of anylength but preferably at least 15 nucleotides, and include asnon-limiting examples, coding and non-coding sequences of a gene, senseand antisense sequences complements, exons, introns, genomic DNA, cDNA,pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinantpolypeptides, isolated and purified naturally occurring DNA or RNAsequences, synthetic RNA and DNA sequences, nucleic acid probes, primersand fragments.

A “fragment” of a polynucleotide sequence provided herein is asubsequence of contiguous nucleotides that is capable of specifichybridization to a target of interest, e.g., a sequence that is at least15 nucleotides in length. The fragments of the invention comprise 15nucleotides, preferably at least 20 nucleotides, more preferably atleast 30 nucleotides, more preferably at least 50 nucleotides, morepreferably at least 50 nucleotides and most preferably at least 60nucleotides of contiguous nucleotides of a polynucleotide of theinvention. A fragment of a polynucleotide sequence can be used inantisense, gene silencing, triple helix or ribozyme technology, or as aprimer, a probe, included in a microarray, or used inpolynucleotide-based selection methods of the invention.

The term “primer” refers to a short polynucleotide, usually having afree 3′ H group, that is hybridized to a template and used for primingpolymerization of a polynucleotide complementary to the target.

The term “probe” refers to a short polynucleotide that is used to detecta polynucleotide sequence, that is complementary to the probe, in ahybridization-based assay. The probe may consist of a “fragment” of apolynucleotide as defined herein.

Polypeptides and fragments

The term “polypeptide”, as used herein, encompasses amino acid chains ofany length but preferably at least 5 amino acids, including full-lengthproteins, in which amino acid residues are linked by covalent peptidebonds. Polypeptides of the present invention may be purified naturalproducts, or may be produced partially or wholly using recombinant orsynthetic techniques. The term may refer to a polypeptide, an aggregateof a polypeptide such as a dimer or other multimer, a fusionpolypeptide, a polypeptide fragment, a polypeptide variant, orderivative thereof.

A “fragment” of a polypeptide is a subsequence of the polypeptide thatperforms a function that is required for the biological activity and/orprovides three dimensional structure of the polypeptide. The term mayrefer to a polypeptide, an aggregate of a polypeptide such as a dimer orother multimer, a fusion polypeptide, a polypeptide fragment, apolypeptide variant, or derivative thereof capable of performing theabove enzymatic activity.

The term “isolated” as applied to the polynucleotide or polypeptidesequences disclosed herein is used to refer to sequences that areremoved from their natural cellular environment. An isolated moleculemay be obtained by any method or combination of methods includingbiochemical, recombinant, and synthetic techniques.

The term “recombinant” refers to a polynucleotide sequence that isremoved from sequences that surround it in its natural context and/or isrecombined with sequences that are not present in its natural context.

A “recombinant” polypeptide sequence is produced by translation from a“recombinant” polynucleotide sequence.

The term “derived from” with respect to polynucleotides and polypeptidesof the invention being “derived from” a particular genera or species,means that the polynucleotide or polypeptide has the same sequence as apolynucleotide or polypeptide found naturally in that genera or species.The polynucleotide or polypeptide which is derived from a genera orspecies may therefore be produced synthetically or recombinantly.

Variants

As used herein, the term “variant” refers to polynucleotide orpolypeptide sequences different from the specifically identifiedsequences, wherein one or more nucleotides or amino acid residues isdeleted, substituted, or added. Variants may be naturally occurringallelic variants, or non-naturally occurring variants. Variants may befrom the same or from other species and may encompass homologues,paralogues and orthologues. In certain embodiments, variants of theinventive polypeptides and polynucleotides possess biological activitiesthat are the same or similar to those of the inventive polypeptides orpolynucleotides. The term “variant” with reference to polypeptides andpolynucleotides encompasses all forms of polypeptides andpolynucleotides as defined herein.

Polynucleotide Variants

Variant polynucleotide sequences preferably exhibit at least 50%, morepreferably at least 51%, more preferably at least 52%, more preferablyat least 53%, more preferably at least 54%, more preferably at least55%, more preferably at least 56%, more preferably at least 57%, morepreferably at least 58%, more preferably at least 59%, more preferablyat least 60%, more preferably at least 61%, more preferably at least62%, more preferably at least 63%, more preferably at least 64%, morepreferably at least 65%, more preferably at least 66%, more preferablyat least 67%, more preferably at least 68%, more preferably at least69%, more preferably at least 70%, more preferably at least 71%, morepreferably at least 72%, more preferably at least 73%, more preferablyat least 74%, more preferably at least 75%, more preferably at least76%, more preferably at least 77%, more preferably at least 78%, morepreferably at least 79%, more preferably at least 80%, more preferablyat least 81%, more preferably at least 82%, more preferably at least83%, more preferably at least 84%, more preferably at least 85%, morepreferably at least 86%, more preferably at least 87%, more preferablyat least 88%, more preferably at least 89%, more preferably at least90%, more preferably at least 91%, more preferably at least 92%, morepreferably at least 93%, more preferably at least 94%, more preferablyat least 95%, more preferably at least 96%, more preferably at least97%, more preferably at least 98%, and most preferably at least 99%identity to a specified polynucleotide sequence. Identity is found overa comparison window of at least 20 nucleotide positions, preferably atleast 50 nucleotide positions, more preferably at least 100 nucleotidepositions, and most preferably over the entire length of the specifiedpolynucleotide sequence.

Polynucleotide sequence identity can be determined in the followingmanner. The subject polynucleotide sequence is compared to a candidatepolynucleotide sequence using BLASTN (from the BLAST suite of programs,version 2.2.5 [Nov 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L.Madden (1999), “Blast 2 sequences—a new tool for comparing protein andnucleotide sequences”, FEMS Microbiol Lett. 174:247-250), which ispublicly available from NCBI (ftp://ftp.ncbi.nih gov/blast/). Thedefault parameters of bl2seq are utilized except that filtering of lowcomplexity parts should be turned off.

The identity of polynucleotide sequences may be examined using thefollowing unix command line parameters:

bl2seq -i nucleotideseq1-j nucleotideseq2 -F F -p blastn

The parameter -F F turns off filtering of low complexity sections. Theparameter -p selects the appropriate algorithm for the pair ofsequences. The bl2seq program reports sequence identity as both thenumber and percentage of identical nucleotides in a line “Identities=”.

Polynucleotide sequence identity may also be calculated over the entirelength of the overlap between a candidate and subject polynucleotidesequences using global sequence alignment programs (e.g. Needleman, S.B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A fullimplementation of the Needleman-Wunsch global alignment algorithm isfound in the needle program in the EMBOSS package (Rice,P. Longden,I.and Bleasby,A. EMBOSS: The European Molecular Biology Open SoftwareSuite, Trends in Genetics June 2000, vol 16, No 6. pp.276-277) which canbe obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. TheEuropean Bioinformatics Institute server also provides the facility toperform EMBOSS-needle global alignments between two sequences on line athttp:/www.ebi.ac.uk/emboss/align/.

Alternatively the GAP program may be used which computes an optimalglobal alignment of two sequences without penalizing terminal gaps. GAPis described in the following paper: Huang, X. (1994) On Global SequenceAlignment. Computer Applications in the Biosciences 10, 227-235.

Use of BLASTN as described above is preferred for use in thedetermination of sequence identity for polynucleotide variants accordingto the present invention.

Polynucleotide variants of the present invention also encompass thosewhich exhibit a similarity to one or more of the specifically identifiedsequences that is likely to preserve the functional equivalence of thosesequences and which could not reasonably be expected to have occurred byrandom chance. Such sequence similarity with respect to polypeptides maybe determined using the publicly available bl2seq program from the BLASTsuite of programs (version 2.2.5 [November 2002]) from NCBI(ftp://ftp.ncbi.nih gov/blast/).

The similarity of polynucleotide sequences may be examined using thefollowing unix command line parameters:

-   -   bl2seq -i nucleotideseq1-j nucleotideseq2-F F -p tblastx

The parameter -F F turns off filtering of low complexity sections. Theparameter -p selects the appropriate algorithm for the pair ofsequences. This program finds regions of similarity between thesequences and for each such region reports an “E value” which is theexpected number of times one could expect to see such a match by chancein a database of a fixed reference size containing random sequences. Thesize of this database is set by default in the bl2seq program.

For small E values, much less than one, the E value is approximately theprobability of such a random match.

Variant polynucleotide sequences preferably exhibit an E value of lessthan 1×10⁻¹⁰ more preferably less than 1×10⁻²⁰, more preferably lessthan 1×10⁻³⁰, more preferably less than 1×10⁴⁰, more preferably lessthan 1×10⁻⁵⁰, more preferably less than 1×10⁻⁶⁰, more preferably lessthan 1×10⁻⁷⁰, more preferably less than 1×10⁻⁸⁰, more preferably lessthan 1×10⁻⁹⁰ and most preferably less than 1×10⁻¹⁰⁰ when compared withany one of the specifically identified sequences.

Alternatively, variant polynucleotides of the present inventionhybridize to a specified polynucleotide sequence, or complements thereofunder stringent conditions.

The term “hybridize under stringent conditions”, and grammaticalequivalents thereof, refers to the ability of a polynucleotide moleculeto hybridize to a target polynucleotide molecule (such as a targetpolynucleotide molecule immobilized on a DNA or RNA blot, such as aSouthern blot or Northern blot) under defined conditions of temperatureand salt concentration. The ability to hybridize under stringenthybridization conditions can be determined by initially hybridizingunder less stringent conditions then increasing the stringency to thedesired stringency.

With respect to polynucleotide molecules greater than about 100 bases inlength, typical stringent hybridization conditions are no more than 25to 30° C. (for example, 10° C.) below the melting temperature (Tm) ofthe native duplex (see generally, Sambrook et al., Eds, 1987, MolecularCloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubelet al., 1987, Current Protocols in Molecular Biology, GreenePublishing,). Tm for polynucleotide molecules greater than about 100bases can be calculated by the formula Tm=81. 5+0.41% (G+C-log (Na+).(Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2ndEd. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390).Typical stringent conditions for polynucleotide of greater than 100bases in length would be hybridization conditions such as prewashing ina solution of 6×SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDSovernight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDSat 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65°C.

With respect to polynucleotide molecules having a length less than 100bases, exemplary stringent hybridization conditions are 5 to 10° C.below Tm. On average, the Tm of a polynucleotide molecule of length lessthan 100 by is reduced by approximately (500/oligonucleotide length)° C.

With respect to the DNA mimics known as peptide nucleic acids (PNAs)(Nielsen et al., Science. 1991 Dec. 6; 254(5037):1497-500) Tm values arehigher than those for DNA-DNA or DNA-RNA hybrids, and can be calculatedusing the formula described in Giesen et al., Nucleic Acids Res. 1998Nov. 1; 26(21):5004-6. Exemplary stringent hybridization conditions fora DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C.below the Tm.

Variant polynucleotides of the present invention also encompassespolynucleotides that differ from the sequences of the invention butthat, as a consequence of the degeneracy of the genetic code, encode apolypeptide having similar activity to a polypeptide encoded by apolynucleotide of the present invention. A sequence alteration that doesnot change the amino acid sequence of the polypeptide is a “silentvariation”. Except for ATG (methionine) and TGG (tryptophan), othercodons for the same amino acid may be changed by art recognizedtechniques, e.g., to optimize codon expression in a particular hostorganism.

Polynucleotide sequence alterations resulting in conservativesubstitutions of one or several amino acids in the encoded polypeptidesequence without significantly altering its biological activity are alsoincluded in the invention. A skilled artisan will be aware of methodsfor making phenotypically silent amino acid substitutions (see, e.g.,Bowie et al., 1990, Science 247, 1306).

Variant polynucleotides due to silent variations and conservativesubstitutions in the encoded polypeptide sequence may be determinedusing the publicly available bl2seq program from the BLAST suite ofprograms (version 2.2.5 [November 2002]) from NCBI (ftp://ftp.ncbi.nihgov/blast/) via the tblastx algorithm as previously described.

Polypeptide Variants

The term “variant” with reference to polypeptides encompasses naturallyoccurring, recombinantly and synthetically produced polypeptides.Variant polypeptide sequences preferably exhibit at least 50%, morepreferably at least 51%, more preferably at least 52%, more preferablyat least 53%, more preferably at least 54%, more preferably at least55%, more preferably at least 56%, more preferably at least 57%, morepreferably at least 58%, more preferably at least 59%, more preferablyat least 60%, more preferably at least 61%, more preferably at least62%, more preferably at least 63%, more preferably at least 64%, morepreferably at least 65%, more preferably at least 66%, more preferablyat least 67%, more preferably at least 68%, more preferably at least69%, more preferably at least 70%, more preferably at least 71%, morepreferably at least 72%, more preferably at least 73%, more preferablyat least 74%, more preferably at least 75%, more preferably at least76%, more preferably at least %, more preferably at least 77%, morepreferably at least 78%, more preferably at least 79%, more preferablyat least 80%, more preferably at least 81%, more preferably at least82%, more preferably at least 83%, more preferably at least 84%, morepreferably at least 85%, more preferably at least 86%, more preferablyat least 87%, more preferably at least 88%, more preferably at least89%, more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, more preferably at least 93%, more preferablyat least 94%, more preferably at least 95%, more preferably at least96%, more preferably at least 97%, more preferably at least 98%, andmost preferably at least 99% identity to a sequences of the presentinvention. Identity is found over a comparison window of at least 20amino acid positions, preferably at least 50 amino acid positions, morepreferably at least 100 amino acid positions, and most preferably overthe entire length of a polypeptide of the invention.

Polypeptide sequence identity can be determined in the following manner.The subject polypeptide sequence is compared to a candidate polypeptidesequence using BLASTP (from the BLAST suite of programs, version 2.2.5[November 2002]) in bl2seq, which is publicly available from NCBI(ftp://ftp.ncbi.nih gov/blast/). The default parameters of bl2seq areutilized except that filtering of low complexity regions should beturned off.

Polypeptide sequence identity may also be calculated over the entirelength of the overlap between a candidate and subject polynucleotidesequences using global sequence alignment programs. EMBOSS-needle(available at http://www.ebi.ac.uk/emboss/align/) and GAP (Huang, X.(1994) On Global Sequence Alignment. Computer Applications in theBiosciences 10, 227-235.) as discussed above are also suitable globalsequence alignment programs for calculating polypeptide sequenceidentity.

Use of BLASTP as described above is preferred for use in thedetermination of polypeptide variants according to the presentinvention.

Polypeptide variants of the present invention also encompass those whichexhibit a similarity to one or more of the specifically identifiedsequences that is likely to preserve the functional equivalence of thosesequences and which could not reasonably be expected to have occurred byrandom chance. Such sequence similarity with respect to polypeptides maybe determined using the publicly available bl2seq program from the BLASTsuite of programs (version 2.2.5 [November 20021) from NCBI(ftp://ftp.ncbi.nih gov/blast/). The similarity of polypeptide sequencesmay be examined using the following unix command line parameters:

-   -   bl2seq -i peptideseq1 -j peptideseq2 -F F -p blastp

Variant polypeptide sequences preferably exhibit an E value of less than1×10⁻¹⁰ more preferably less than 1×10⁻²⁰, more preferably less than1×10⁻³⁰, more preferably less than 1×10⁴⁰, more preferably less than1×10⁻⁵⁰, more preferably less than 1×10⁻⁶⁰, more preferably less than1×10⁻⁷⁰, more preferably less than 1×10⁻⁸⁰, more preferably less than1×10⁻⁹⁰ and most preferably less than 1×10⁻¹⁰⁰ when compared with anyone of the specifically identified sequences.

The parameter -F F turns off filtering of low complexity sections. Theparameter -p selects the appropriate algorithm for the pair ofsequences. This program finds regions of similarity between thesequences and for each such region reports an “E value” which is theexpected number of times one could expect to see such a match by chancein a database of a fixed reference size containing random sequences. Forsmall E values, much less than one, this is approximately theprobability of such a random match.

Conservative substitutions of one or several amino acids of a describedpolypeptide sequence without significantly altering its biologicalactivity are also included in the invention. A skilled artisan will beaware of methods for making phenotypically silent amino acidsubstitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

Constructs, Vectors and Components Thereof

The term “genetic construct” refers to a polynucleotide molecule,usually double-stranded DNA, which may have inserted into it anotherpolynucleotide molecule (the insert polynucleotide molecule) such as,but not limited to, a cDNA molecule. A genetic construct may contain thenecessary elements that permit transcribing the insert polynucleotidemolecule, and, optionally, translating the transcript into apolypeptide. The insert polynucleotide molecule may be derived from thehost cell, or may be derived from a different cell or organism and/ormay be a recombinant polynucleotide. Once inside the host cell thegenetic construct may become integrated in the host chromosomal DNA. Thegenetic construct may be linked to a vector.

The term “vector” refers to a polynucleotide molecule, usually doublestranded DNA, which is used to transport the genetic construct into ahost cell. The vector may be capable of replication in at least oneadditional host system, such as E. coli.

The term “expression construct” refers to a genetic construct thatincludes the necessary elements that permit transcribing the insertpolynucleotide molecule, and, optionally, translating the transcriptinto a polypeptide. An expression construct typically comprises in a 5′to 3′ direction:

-   -   a) a promoter functional in the host cell into which the        construct will be transformed,    -   b) the polynucleotide to be expressed, and    -   c) a terminator functional in the host cell into which the        construct will be transformed.

The term “coding region” or “open reading frame” (ORF) refers to thesense strand of a genomic DNA sequence or a cDNA sequence that iscapable of producing a transcription product and/or a polypeptide underthe control of appropriate regulatory sequences. The coding sequence isidentified by the presence of a 5′ translation start codon and a 3′translation stop codon. When inserted into a genetic construct, a“coding sequence” is capable of being expressed when it is operablylinked to promoter and terminator sequences.

“Operably-linked” means that the sequenced to be expressed is placedunder the control of regulatory elements that include promoters,tissue-specific regulatory elements, temporal regulatory elements,enhancers, repressors and terminators.

The term “noncoding region” refers to untranslated sequences that areupstream of the translational start site and downstream of thetranslational stop site. These sequences are also referred torespectively as the 5′ UTR and the 3′ UTR. These regions includeelements required for transcription initiation and termination and forregulation of translation efficiency.

Terminators are sequences, which terminate transcription, and are foundin the 3′ untranslated ends of genes downstream of the translatedsequence. Terminators are important determinants of mRNA stability andin some cases have been found to have spatial regulatory functions.

The term “promoter” refers to nontranscribed cis-regulatory elementsupstream of the coding region that regulate gene transcription.Promoters comprise cis-initiator elements which specify thetranscription initiation site and conserved boxes such as the TATA box,and motifs that are bound by transcription factors.

A “transgene” is a polynucleotide that is taken from one organism andintroduced into a different organism by transformation. The transgenemay be derived from the same species or from a different species as thespecies of the organism into which the transgene is introduced.

An “inverted repeat” is a sequence that is repeated, where the secondhalf of the repeat is in the complementary strand, e.g.,

(5′)GATCTA . . . TAGATC(3′) (3′)CTAGAT . . . ATCTAG(5′)

Read-through transcription will produce a transcript that undergoescomplementary base-pairing to form a hairpin structure provided thatthere is a 3-5 by spacer between the repeated regions.

A “transgenic plant” refers to a plant which contains new geneticmaterial as a result of genetic manipulation or transformation. The newgenetic material may be derived from a plant of the same species as theresulting transgenic plant or from a different species.

The terms “to alter expression of” and “altered expression” of apolynucleotide or polypeptide of the invention, are intended toencompass the situation where genomic DNA corresponding to apolynucleotide of the invention is modified thus leading to alteredexpression of a polynucleotide or polypeptide of the invention.Modification of the genomic DNA may be through genetic transformation orother methods known in the art for inducing mutations. The “alteredexpression” can be related to an increase or decrease in the amount ofmessenger RNA and/or polypeptide produced and may also result in alteredactivity of a polypeptide due to alterations in the sequence of apolynucleotide and polypeptide produced.

The term “biomass” refers to the size and/or mass and/or number ofvegetative organs of the plant at a particular age or developmentalstage. Thus a plant with increased biomass has increased size and/ormass and/or number of vegetative organs than a suitable control plant ofthe same age or at an equivalent developmental stage. Conversely, aplant with decreased biomass has decreased size and/or mass and/ornumber of vegetative organs than a suitable control. Altered biomass mayalso involve an alteration in rate of growth and/or rate of formation ofvegetative organs during some or all periods of the life cycle of aplant relative to a suitable control. Thus altered biomass may result inan advance or delay in the time taken for such a plant to reach acertain developmental stage.

Suitable control plants may include non-transformed plants of the samespecies and variety, or plants of the same species or varietytransformed with a control construct.

The invention provides methods for producing and selecting plants withaltered biomass relative to suitable control plants, including plantswith both increased and decreased biomass and plants produced by suchmethods.

The invention provides a polynucleotide (SEQ ID NO:10) encoding apolypeptide (SEQ ID NO:1) which modulates biomass in plants. Theinvention provides polynucleotide variants of SEQ ID NO:10 (SEQ ID NOs:11 to 18) which encode polypeptide variants of SEQ ID NO:1 (SEQ ID NO:2to 9). The applicants have also identified a consensus polypeptidesequence motif present in SEQ ID NO:1 and all of the polypeptidevariants of SEQ ID NO:1, as shown in SEQ ID NO:20, and a furtherconsensus motif (SEQ ID NO: 21) present in SEQ ID NO:1 (ORF54) and allpolypeptide variants thereof that are derived from dicotyledonousplants.

Methods for Isolating Polynucleotides

The polynucleotide molecules of the invention can be isolated by using avariety of techniques known to those of ordinary skill in the art. Byway of example, such polypeptides can be isolated through use of thepolymerase chain reaction (PCR) described in Mullis et al., Eds. 1994The Polymerase Chain Reaction, Birkhauser, incorporated herein byreference. The polypeptides of the invention can be amplified usingprimers, as defined herein, derived from the polynucleotide sequences ofthe invention.

Further methods for isolating polynucleotides of the invention, orpolynucleotides useful in methods of the invention, include use of all,or portions of, the polynucleotides set forth herein as hybridizationprobes. The technique of hybridizing labelled polynucleotide probes topolynucleotides immobilized on solid supports such as nitrocellulosefilters or nylon membranes, can be used to screen the genomic or cDNAlibraries. Exemplary hybridization and wash conditions are:hybridization for 20 hours at 65° C. in 5.0×SSC, 0.5% sodium dodecylsulfate, 1× Denhardt's solution; washing (three washes of twenty minuteseach at 55° C.) in 1.0×SSC, 1% (w/v) sodium dodecyl sulfate, andoptionally one wash (for twenty minutes) in 0.5×SSC, 1% (w/v) sodiumdodecyl sulfate, at 60° C. An optional further wash (for twenty minutes)can be conducted under conditions of 0.1×SSC, 1% (w/v) sodium dodecylsulfate, at 60° C.

The polynucleotide fragments of the invention may be produced bytechniques well-known in the art such as restriction endonucleasedigestion and oligonucleotide synthesis.

A partial polynucleotide sequence may be used, in methods well-known inthe art to identify the corresponding full-length polynucleotidesequence. Such methods include PCR-based methods, 5′RACE (Frohman Mass.,1993, Methods Enzymol. 218: 340-56) and hybridization-based method,computer/database -based methods. Further, by way of example, inversePCR permits acquisition of unknown sequences, flanking thepolynucleotide sequences disclosed herein, starting with primers basedon a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186,incorporated herein by reference). The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template. Divergent primers are designed from the known region. Inorder to physically assemble full-length clones, standard molecularbiology approaches can be utilized (Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).

It may be beneficial, when producing a transgenic plant from aparticular species, to transform such a plant with a sequence orsequences derived from that species. The benefit may be to alleviatepublic concerns regarding cross-species transformation in generatingtransgenic organisms. Additionally when down-regulation of a gene is thedesired result, it may be necessary to utilise a sequence identical (orat least highly similar) to that in the plant, for which reducedexpression is desired. For these reasons among others, it is desirableto be able to identify and isolate orthologues of a particular gene inseveral different plant species. Variants (including orthologues) may beidentified by the methods described.

Methods for Identifying Variants

Physical Methods

Variant polynucleotides may be identified using PCR-based methods(Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser).Typically, the polynucleotide sequence of a primer, useful to amplifyvariant polynucleotide molecules by PCR, may be based on a sequenceencoding a conserved region of the corresponding amino acid sequence.

Alternatively library screening methods will be known to those skilledin the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd. Cold Spring Harbor Press, 1987) may be employed. When identifyingvariants of the probe sequence hybridisation and/or wash stringencyconditions will typically be reduced relative to when exact sequencematches are sought.

Polypeptide variants of the invention may be identified by physicalmethods, for example by screening expression libraries using antibodiesraised against polypeptides of the invention (Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) orby identifying polypeptides from natural sources with the aid of suchantibodies.

Computer Based Methods

The variant sequences of the invention, including both polynucleotideand polypeptide variants, may also be identified by computer-basedmethods well-known to those skilled in the art, using public domainsequence alignment algorithms and sequence similarity search tools tosearch sequence databases (public domain databases include Genbank,EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29:1-10 and 11-16, 2001 for examples of online resources. Similaritysearches retrieve and align target sequences for comparison with asequence to be analyzed (i.e., a query sequence). Sequence comparisonalgorithms use scoring matrices to assign an overall score to each ofthe alignments.

An exemplary family of programs useful for identifying variants insequence databases is the BLAST suite of programs (version 2.2.5 [Nov2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which arepublicly available from (ftp://ftp.ncbi.nih gov/blast/) or from theNational Center for Biotechnology Information (NCBI), National Libraryof Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894 USA. The NCBIserver also provides the facility to use the programs to screen a numberof publicly available sequence databases. BLASTN compares a nucleotidequery sequence against a nucleotide sequence database. BLASTP comparesan amino acid query sequence against a protein sequence database. BLASTXcompares a nucleotide query sequence translated in all reading framesagainst a protein sequence database. tBLASTN compares a protein querysequence against a nucleotide sequence database dynamically translatedin all reading frames. tBLASTX compares the six-frame translations of anucleotide query sequence against the six-frame translations of anucleotide sequence database. The BLAST programs may be used withdefault parameters or the parameters may be altered as required torefine the screen.

The use of the BLAST family of algorithms, including BLASTN, BLASTP, andBLASTX, is described in the publication of Altschul et al., NucleicAcids Res. 25: 3389-3402, 1997.

The “hits” to one or more database sequences by a queried sequenceproduced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similaralgorithm, align and identify similar portions of sequences. The hitsare arranged in order of the degree of similarity and the length ofsequence overlap. Hits to a database sequence generally represent anoverlap over only a fraction of the sequence length of the queriedsequence.

The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce“Expect” values for alignments. The Expect value (E) indicates thenumber of hits one can “expect” to see by chance when searching adatabase of the same size containing random contiguous sequences. TheExpect value is used as a significance threshold for determining whetherthe hit to a database indicates true similarity. For example, an E valueof 0.1 assigned to a polynucleotide hit is interpreted as meaning thatin a database of the size of the database screened, one might expect tosee 0.1 matches over the aligned portion of the sequence with a similarscore simply by chance. For sequences having an E value of 0.01 or lessover aligned and matched portions, the probability of finding a match bychance in that database is 1% or less using the BLASTN, BLASTP, BLASTX,tBLASTN or tBLASTX algorithm.

Multiple sequence alignments of a group of related sequences can becarried out with CLUSTALW (Thompson, J. D., Higgins, D. G. and Gibson,T. J. (1994) CLUSTALW: improving the sensitivity of progressive multiplesequence alignment through sequence weighting, positions-specific gappenalties and weight matrix choice. Nucleic Acids Research,22:4673-4680, http://www-igbmc.u-strasbg.fr/Biolnfo/ClustalW/Top.html)or T-COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Heringa,T-Coffee: A novel method for fast and accurate multiple sequencealignment, J. Mol. Biol. (2000) 302: 205-217))or PILEUP, which usesprogressive, pairwise alignments. (Feng and Doolittle, 1987, J. Mol.Evol. 25, 351).

Pattern recognition software applications are available for findingmotifs or signature sequences. For example, MEME (Multiple Em for MotifElicitation) finds motifs and signature sequences in a set of sequences,and MAST (Motif Alignment and Search Tool) uses these motifs to identifysimilar or the same motifs in query sequences. The MAST results areprovided as a series of alignments with appropriate statistical data anda visual overview of the motifs found. MEME and MAST were developed atthe University of California, San Diego.

PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmannet al., 1999, Nucleic Acids Res. 27, 215) is a method of identifying thefunctions of uncharacterized proteins translated from genomic or cDNAsequences. The PROSITE database (www.expasy.org/prosite) containsbiologically significant patterns and profiles and is designed so thatit can be used with appropriate computational tools to assign a newsequence to a known family of proteins or to determine which knowndomain(s) are present in the sequence (Falquet et al., 2002, NucleicAcids Res. 30, 235). Prosearch is a tool that can search SWISS-PROT andEMBL databases with a given sequence pattern or signature.

Methods for Isolating Polypeptides

The polypeptides of the invention, including variant polypeptides, maybe prepared using peptide synthesis methods well known in the art suchas direct peptide synthesis using solid phase techniques (e.g. Stewartet al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, SanFrancisco Calif., or automated synthesis, for example using an AppliedBiosystems 431A Peptide Synthesizer (Foster City, Calif.). Mutated formsof the polypeptides may also be produced during such syntheses.

The polypeptides and variant polypeptides of the invention may also bepurified from natural sources using a variety of techniques that arewell known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology,Vol. 182, Guide to Protein Purification).

Alternatively the polypeptides and variant polypeptides of the inventionmay be expressed recombinantly in suitable host cells and separated fromthe cells as discussed below.

Methods for producing constructs and vectors

The genetic constructs of the present invention comprise one or morepolynucleotide sequences of the invention and/or polynycleotidesencoding polypeptides of the invention, and may be useful fortransforming, for example, bacterial, fungal, insect, mammalian or plantorganisms. The genetic constructs of the invention are intended toinclude expression constructs as herein defined.

Methods for producing and using genetic constructs and vectors are wellknown in the art and are described generally in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring HarborPress, 1987; Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing, 1987).

Methods for Producing Host Cells Comprising Constructs and Vectors

The invention provides a host cell which comprises a genetic constructor vector of the invention. Host cells may be derived from, for example,bacterial, fungal, insect, mammalian or plant organisms.

Host cells comprising genetic constructs, such as expression constructs,of the invention are useful in methods well known in the art (e.g.Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd Ed. ColdSpring Harbor Press, 1987 ; Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing, 1987) for recombinant productionof polypeptides of the invention. Such methods may involve the cultureof host cells in an appropriate medium in conditions suitable for orconducive to expression of a polypeptide of the invention. The expressedrecombinant polypeptide, which may optionally be secreted into theculture, may then be separated from the medium, host cells or culturemedium by methods well known in the art (e.g. Deutscher, Ed, 1990,Methods in Enzymology, Vol 182, Guide to Protein Purification).

Host cells of the invention may also be useful in methods for productionof an enzymatic product generated by an expressed polypeptide of theinvention. Such methods may involve culturing the host cells of theinvention in a medium suitable for expression of a recombinantpolypeptide of the invention, optionally in the presence of additionalenzymatic substrate for the expressed polypeptide of the invention. Theenzymatic product produced may then be separated from the host cells ormedium by a variety of art standard methods.

Methods for Producing Plant Cells and Plants Comprising Constructs andVectors

The invention further provides plant cells which comprise a geneticconstruct of the invention, and plant cells modified to alter expressionof a polynucleotide or polypeptide of the invention. Plants comprisingsuch cells also form an aspect of the invention.

Production of plants altered in biomass may be achieved through methodsof the invention. Such methods may involve the transformation of plantcells and plants, with a construct of the invention designed to alterexpression of a polynucleotide or polypeptide capable of modulatingbiomass production in such plant cells and plants. Such methods alsoinclude the transformation of plant cells and plants with a combinationof the construct of the invention and one or more other constructsdesigned to alter expression of one or more polypeptides or polypeptidescapable of modulating biomass production in such plant cells and plants.

Methods for transforming plant cells, plants and portions thereof withpolynucleotides are described in Draper et al., 1988, Plant GeneticTransformation and Gene Expression. A Laboratory Manual, Blackwell Sci.Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer toPlants. Springer-Verlag, Berlin.; and Gelvin et al., 1993, PlantMolecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review oftransgenic plants, including transformation techniques, is provided inGalun and Breiman, 1997, Transgenic Plants. Imperial College Press,London.

Methods for Genetic Manipulation of Plants

A number of strategies for genetically manipulating plants are available(e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297). Forexample, strategies may be designed to increase expression of apolynucleotide/polypeptide in a plant cell, organ and/or at a particulardevelopmental stage where/when it is normally expressed or toectopically express a polynucleotide/polypeptide in a cell, tissue,organ and/or at a particular developmental stage which/when it is notnormally expressed. The expressed polynucleotide/polypeptide may bederived from the plant species to be transformed or may be derived froma different plant species.

Transformation strategies may be designed to reduce expression of apolynucleotide/polypeptide in a plant cell, tissue, organ or at aparticular developmental stage which/when it is normally expressed. Suchstrategies are known as gene silencing strategies.

Genetic constructs for expression of genes in transgenic plantstypically include promoters for driving the expression of one or morecloned polynucleotide, terminators and selectable marker sequences todetest presence of the genetic construct in the transformed plant.

The promoters suitable for use in the constructs of this invention arefunctional in a cell, tissue or organ of a monocot or dicot plant andinclude cell-, tissue- and organ-specific promoters, cell cycle specificpromoters, temporal promoters, inducible promoters, constitutivepromoters that are active in most plant tissues, and recombinantpromoters. Choice of promoter will depend upon the temporal and spatialexpression of the cloned polynucleotide, so desired. The promoters maybe those normally associated with a transgene of interest, or promoterswhich are derived from genes of other plants, viruses, and plantpathogenic bacteria and fungi. Those skilled in the art will, withoutundue experimentation, be able to select promoters that are suitable foruse in modifying and modulating plant traits using genetic constructscomprising the polynucleotide sequences of the invention. Examples ofconstitutive plant promoters include the CaMV 35S promoter, the nopalinesynthase promoter and the octopine synthase promoter, and the Ubi 1promoter from maize. Plant promoters which are active in specifictissues, respond to internal developmental signals or external abioticor biotic stresses are described in the scientific literature. Exemplarypromoters are described, e.g., in WO 02/00894, which is hereinincorporated by reference.

Exemplary terminators that are commonly used in plant transformationgenetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35Sterminator, the Agrobacterium tumefaciens nopaline synthase or octopinesynthase terminators, the Zea mays zein gene terminator, the Oryzasativa ADP-glucose pyrophosphorylase terminator and the Solanumtuberosum PI-II terminator.

Selectable markers commonly used in plant transformation include theneomycin phophotransferase II gene (NPT II) which confers kanamycinresistance, the aadA gene, which confers spectinomycin and streptomycinresistance, the phosphinothricin acetyl transferase (bar gene) forIgnite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycinphosphotransferase gene (hpt) for hygromycin resistance.

Use of genetic constructs comprising reporter genes (coding sequenceswhich express an activity that is foreign to the host, usually anenzymatic activity and/or a visible signal (e.g., luciferase, GUS, GFP)which may be used for promoter expression analysis in plants and planttissues are also contemplated. The reporter gene literature is reviewedin Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995,In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) SpringerVerlag. Berline, pp. 325-336.

Gene silencing strategies may be focused on the gene itself orregulatory elements which effect expression of the encoded polypeptide.“Regulatory elements” is used here in the widest possible sense andincludes other genes which interact with the gene of interest.

Genetic constructs designed to decrease or silence the expression of apolynucleotide/polypeptide of the invention may include an antisensecopy of a polynucleotide of the invention. In such constructs thepolynucleotide is placed in an antisense orientation with respect to thepromoter and terminator.

An “antisense” polynucleotide is obtained by inverting a polynucleotideor a segment of the polynucleotide so that the transcript produced willbe complementary to the mRNA transcript of the gene, e.g.,

5′GATCTA 3′ (coding 3′CTAGAT 5′ (antisense strand) strand) 3′CUAGAU 5′mRNA 5′GAUCUCG 3′ antisense RNA

Genetic constructs designed for gene silencing may also include aninverted repeat. An ‘inverted repeat’ is a sequence that is repeatedwhere the second half of the repeat is in the complementary strand,e.g.,

5′-GATCTA . . . TAGATC-3′ 3′-CTAGAT . . . ATCTAG-5′

The transcript formed may undergo complementary base pairing to form ahairpin structure. Usually a spacer of at least 3-5 by between therepeated region is required to allow hairpin formation.

Another silencing approach involves the use of a small antisense RNAtargeted to the transcript equivalent to an miRNA (Llave et al., 2002,Science 297, 2053). Use of such small antisense RNA corresponding topolynucleotide of the invention is expressly contemplated.

The term genetic construct as used herein also includes small antisenseRNAs and other such polynucleotides useful for effecting gene silencing.

Transformation with an expression construct, as herein defined, may alsoresult in gene silencing through a process known as sense suppression(e.g. Napoli et al., 1990, Plant Cell 2, 279; de Carvalho Niebel et al.,1995, Plant Cell, 7, 347). In some cases sense suppression may involveover-expression of the whole or a partial coding sequence but may alsoinvolve expression of non-coding region of the gene, such as an intronor a 5′ or 3′ untranslated region (UTR). Chimeric partial senseconstructs can be used to coordinately silence multiple genes (Abbott etal., 2002, Plant Physiol. 128(3): 844-53; Jones et al., 1998, Planta204: 499-505). The use of such sense suppression strategies to silencethe expression of a polynucleotide of the invention is alsocontemplated.

The polynucleotide inserts in genetic constructs designed for genesilencing may correspond to coding sequence and/or non-coding sequence,such as promoter and/or intron and/or 5′ or 3′ UTR sequence, or thecorresponding gene.

Other gene silencing strategies include dominant negative approaches andthe use of ribozyme constructs (McIntyre, 1996, Transgenic Res, 5, 257).

Pre-transcriptional silencing may be brought about through mutation ofthe gene itself or its regulatory elements. Such mutations may includepoint mutations, frameshifts, insertions, deletions and substitutions.

The following are representative publications disclosing genetictransformation protocols that can be used to genetically transform thefollowing plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18,572); maize (U.S. Pat. Nos 5,177,010 and 5,981,840); wheat (Ortiz etal., 1996, Plant Cell Rep. 15, 1996, 877); tomato (U.S. Pat. No.5,159,135); potato (Kumar et al., 1996 Plant J. 9, : 821); cassava (Liet al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al.,1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science227, 1229); cotton (U.S. Pat. Nos. 5,846,797 and 5,004,863); grasses(U.S. Pat. Nos. 5,187,073, 6,020,539); peppermint (Niu et al., 1998,Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, PlantSci.104, 183); caraway (Krens et al., 1997, Plant Cell Rep, 17, 39);banana (U.S. Pat. No. 5,792,935); soybean (U.S. Pat. Nos. 5,416,011;5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat.No. 5,952,543); poplar (U.S. Pat. No. 4,795,855); monocots in general(U.S. Pat. Nos. 5,591,616 and 6,037,522); brassica (U.S. Pat. Nos.5,188,958; 5,463,174 and 5,750,871); alfalfa (Weeks et al., (2008)Transgenic Research 17: 587-597; Samac et al., (2006) Methods Mol Biol,Vol 343, Agrobacterium Protocols. 2nd edition. Totowa, N.J.: HumanaPress. p 301-311.); and cereals (U.S. Pat. No. 6,074,877). Other speciesare contemplated and suitable methods and protocols are available in thescientific literature for use by those skilled in the art.

Several further methods known in the art may be employed to alterexpression of a nucleotide and/or alter expression or activity of apolypeptide of the invention, or used in a method of the invention. Suchmethods include but are not limited to Tilling (Till et al., 2003,Methods Mol Biol, 2%, 205), so called “Deletagene” technology (Li etal., 2001, Plant Journal 27(3), 235) and the use of artificialtranscription factors such as synthetic zinc finger transcriptionfactors. (e.g. Jouvenot et al., 2003, Gene Therapy 10, 513).Additionally antibodies or fragments thereof, targeted to a particularpolypeptide may also be expressed in plants to modulate the activity ofthat polypeptide (Jobling et al., 2003, Nat. Biotechnol., 21(1), 35).Transposon tagging approaches may also be applied. Additionally peptidesinteracting with a polypeptide of the invention may be identifiedthrough technologies such as phage-display (Dyax Corporation). Suchinteracting peptides may be expressed in or applied to a plant to affectactivity of a polypeptide of the invention. Plantibodies (Stoger et al.,Current Opinion in Biotechnology Volume 13, Issue 2, 1 April 2002, Pages161-166; Sudarshana et al., Methods Mol Biol. 2007;354:183-95.) may alsobe used to modulate expression or activity of a polypeptide in a plant.Use of each of the above approaches, including the silencing methodsdiscussed, in alteration of expression of a nucleotide and/or expressionor activity of a polypeptide of the invention is specificallycontemplated.

Methods for Selecting Plants

Methods are also provided for selecting plants with altered biomass.Such methods involve testing of plants for altered for the expression ofa polynucleotide or polypeptide of the invention. Such methods may beapplied at a young age or early developmental stage when the alteredbiomass may not necessarily be visible, to accelerate breeding programsdirected toward improving biomass.

The expression of a polynucleotide, such as a messenger RNA, is oftenused as an indicator of expression of a corresponding polypeptide.Exemplary methods for measuring the expression of a polynucleotideinclude but are not limited to Northern analysis, RT-PCR and dot-blotanalysis (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd. Cold Spring Harbor Press, 1987). Polynucleotides or portions of thepolynucleotides of the invention are thus useful as probes or primers,as herein defined, in methods for the identification of plants withaltered biomass. The polypeptides of the invention may be used as probesin hybridization experiments, or as primers in PCR based experiments,designed to identify such plants.

Alternatively antibodies may be raised against polypeptides of theinvention. Methods for raising and using antibodies are standard in theart (see for example: Antibodies, A Laboratory Manual, Harlow A Lane,Eds, Cold Spring Harbour Laboratory, 1998). Such antibodies may be usedin methods to detect altered expression of polypeptides which modulatebiomass in plants. Such methods may include ELISA (Kemeny, 1991, APractical Guide to ELISA, NY Pergamon Press) and Western analysis(Towbin & Gordon, 1994, J Immunol Methods, 72, 313).

These approaches for analysis of polynucleotide or polypeptideexpression and the selection of plants with altered expression areuseful in conventional breeding programs designed to produce varietieswith altered biomass.

Plants

The plants of the invention may be grown and either self-ed or crossedwith a different plant strain and the resulting hybrids, with thedesired phenotypic characteristics, may be identified. Two or moregenerations may be grown to ensure that the subject phenotypiccharacteristics are stably maintained and inherited. Plants resultingfrom such standard breeding approaches also form an aspect of thepresent invention.

It may be desirable to either increase or decrease biomass in aparticular plant species. Increased biomass would be advantageous forexample in human food, forage and forestry crops as well as inornamental plants. Decreased biomass may also be desirable in certain ofthe above cases, for example in the miniaturization of ornamentalplants.

Biomass in a plant may also be altered through methods of the invention.Such methods may involve the transformation of plant cells and plants,with a construct of the invention designed to alter expression of apolynucleotide or polypeptide which modulates biomass in such plantcells and plants. Such methods also include the transformation of plantcells and plants with a combination of the construct of the inventionand one or more other constructs designed to alter expression of one ormore polynucleotides or polypeptides which modulates biomass in plants.

Exemplary methods for assessing growth rate and biomass in plants of theinvention are provided in Boyes D C et al., 2001, Plant Cell.13(7):1499-510; Lancashire P.D et al., 1991, Ann Appl. Biol. 119:560-601, and in Example 1 below.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood with reference to theaccompanying drawings in which:

FIG. 1 shows the summary output of a BLAST-P search of the NR-PLANTdatabase (release date 1 Jan. 2005) in which the ORF54 polypeptide (SEQID NO:1) was used as a seed sequence.

FIG. 2-1 through 2-4 shows PrettyPlot alignment of polypeptides (SEQ IDNO: 1 to 9), including ORF54 and variants thereof

FIG. 3-1 through 3-4 shows a “T-COFFEE” alignment of the seven repeatelements, termed RCC repeats, found in the polypeptide sequences (SEQ IDNO: 1 to 9) as used by the applicants to identify a consensus region(SEQ ID NO: 10) present in each repeat of all the sequences.

FIG. 4 shows a map of a vector, for plant transformation, comprisingORF54 cloned in anti-sense orientation relative to the CaMV 35Spromoter. The sequence of the vector is represented in SEQ ID NO:20.

FIG. 5 shows a DNA gel-blot analysis on genomic DNA from ORF54 T0transgenic plants digested with HindIII and probed with a fragment ofORF54 coding sequence to determine gene copy number and to identifyindependent transformation events.

FIG. 5 a-5 e show growth parameters observed for these ORF54 T1 plantlines compared to the best performing wild type control (Nipponbare) intwo separate experiments. Where FIG. 5 a Plant height measurements fromExperiment 1, FIG. 5 b Plant tiller number from Experiment 1, FIG. 5 cTiller number in ORF54 lines that exceeded the tittering capacity in thebest performing wild-type control (Nipponbare), FIG. 5 d Plant heightmeasurements from Experiment 2 and FIG. 5 e Plant tiller number fromExperiment 2.

FIG. 6 a shows shoot biomass analysis between ORF54 plants and wild-typecontrol (Nipponbare). Student's t-test: *1 Mean differences betweensample and Nipponbare is highly significant (p-0.01). *2 Meandifferences between sample and Nipponbare is very highly significant(p=0.001).

FIG. 6 b shows dry matter yield in ORF54 lines that exceeded the drymatter yielding capacity in the best performing wild-type control(Nipponbare).

FIG. 7-1 through 7-4 shows an alignment of the ORF54 polypeptide andvariants thereof from monocotyledonous species. Highlighting shows theposition a consensus sequence motif (SEQ ID NO: 20) completely conservedin all of the sequences.

FIG. 8-1 through 8-2 shows an alignment of the ORF54 polypeptide andvariants thereof from dicotyledonous species. Highlighting shows theposition a consensus sequence motif (SEQ ID NO: 21) completely conservedin all of the sequences.

FIG. 9 shows altered biomass (dry weight in grams) in the transgenicryegrass lines (1V20, 1V5, 1V1, 1V8, 1V11, 1V3, 2V9, 2V8, 2V5, 2V7, 2V2,3V5, 3V12, 3V11, 3V10 and 3V7) over the non-transgenic control lines(T40, T41 and T101) bars over the columns represent standard deviation.

EXAMPLES

The invention will now be illustrated with reference to the followingnon-limiting examples.

Example 1 Increased Biomass by Down Regulation of a Polynucleotide ofthe Invention in Transgenic Plants

ORF54

A polynucleotide sequence designated ORF54 (SEQ ID NO: 10), encoding thepolypeptide of SEQ ID NO:1, was identified in a ViaLactia BiosciencesLtd proprietary ryegrass (Lolium perenne) GeneThresher (Orion Genomics)genomic library.

ORF54 Variants

The polypeptide sequence of ORF54 (SEQ ID NO:1) was used as a seedsequence to perform a BLASTP search against the NR-Plant database(release date 2005-01-01) to identify variants of ORF54. A cut-off valueof less than or equal to le-140 was identified as distinguishing ORF54variants based upon the applicant's assessment of the associated scorevalue and annotations in the public data base. The BLASTP output summaryis shown in FIG. 1.

The selected variant sequences were aligned using the EMBOSS tool EMMA(Thompson et al 1994), which is an interface to the popular multiplealignment program ClustalW. Aligned sequences were visualised usinganother EMBOSS tool called prettyplot as shown in FIG. 2.

The polypeptide sequences of ORF54 variants are listed as SEQ ID NO:2 to9 in the sequence listing. The corresponding polynucleotide sequencesare listed as SEQ ID NO:11 to 18 respectively.

Analysis of the polypeptide sequences revealed the prescence of sevenrepeats per polypeptide sequence and the repeat sequences are termed RCCrepeats. The repeat sequences from all polypeptides (SEQ ID NO:1-9)above were aligned using the T-COFFEE alignment programmeVersion_(—)1.37 (Notredame et. al 2000, Higgins) and the outcome ispresented in FIG. 3.

A polypeptide motif based on, and present in, all of the RCC repeatsfrom ORF54 and each of the ORF54 variants was identified and isrepresented in FIG. 3.

A Vector Comprising ORF54

The ORF54 polynucleotide sequence (SEQ ID NO:10) was cloned in theanti-sense orientation upstream of the double 35S promoter in order todown-regulate expression of the ORF54 homologue in rice. The vectorcomprising ORF54 was produced by standard molecular biology techniques.A map of the binary vector is shown in FIG. 4. The sequence of thevector is represented in SEQ ID NO:19.

Plant Transformation—Rice

The purified binary vector (SEQ ID NO:19) was introduced intoAgrobacterium strain EHA105 by electroporation (den Dulk-Ras A andHooykaas P J.) and the suspension was incubated at 26° C. for 30minutes. A small aliquot was plated on AB minimal medium(Schmidt-Eisenlohr et. al 1999) containing Kanamycin at 100 mg/L. Plateswere incubated at 26° C. for 3 days and single colonies were tested forpresence of the plasmid using construct specific primers andtransformation confirmed.

Agrobacterium

cultures were grown in AG minimal medium containing 100 mg/L kanamycinat 26° C. with shaking (200 rpm). The Agrobacterium suspensions werepelleted at 5,000 rpm for 5 minutes, washed once in basal MS mediumcontaining 1% glucose and 3% sucrose, pH 5.2, and re-suspended in samemedium containing 200 μM acetosyringone to OD₆₀₀ 0.6-0.8.

A. tumefaciens containing the binary vector ORF54 were used toco-cultivate at least 1,000 immature rice (Oryza sativa) cv. Nipponbareembryos. Immature seeds from rice were washed in sterile water and thensurface sterilized with sodium hypochlorite containing 1.25% activechlorine with 10 μL Tween 20 for 20 minutes. After sterilization, theseeds were washed several times with sterile water and blotted dry onsterile filter paper (3M). The seeds were de-husked manually usingsterile pair of forceps and the embryo dissected out with sterile knife.The isolated embryos were immersed in Agrobacterium suspension for 30minutes with continuous shaking at 100 rpm in a 10 mL culture tube. Theexcess liquid was drained off and the embryos blotted on to sterilefilter paper before placing them on to co-cultivation medium containingMS medium (Murashige and Skoog, 1964) supplemented with 3% sucrose, 1%glucose, 2 mg/L 2,4-D, 0.1 mg/L BA, 400 μM acetosyringone, pH 5.2 for 4days in dark. After co-cultivation, the calli forming embryos weresub-cultured once every two weeks on selection medium consisting of MSmedium supplemented with 3% sucrose, 1% glucose, 2 mg/L 2,4-D(2,4-dichlorophenoxy acetic acid), 0.1 mg/L BA (benzyl adenine) andcontaining 50 mg/L hygromycin and 300 mg/L timentinTM (ticarcillin+clavulanic acid) till at-least 30 healthy calli showing green spotsindicative of healthy shoot emergence was achieved. Calli containing thegreen spots were transferred to selection medium lacking 2,4-D toregenerate a minimum of 10 transformed plants. Regenerated plants wererooted and then transplanted to six inch pots containing soil and plantsgrown in greenhouse. DNA gel-blot analysis was carried out (FIG. 5) bydigesting genomic DNA from transgenic plants with HindIll and probingwith a fragment of ORF54 coding sequence to determine gene copy numberand to identify five independent transformation events. T1 seeds wereharvested from the transformed plants (T0).

T1 Plant Phenotyping

Thirty seeds from Southern positive T0 plants were sown in individualcups containing cocopeat and twenty healthy plants out of them weretransplanted in the green house. These plants were arranged using a CRDusing the random numbers from a random table.

T1 plant phenotyping was carried out in two separate experiments. Thefirst experiment involved progeny lines from T0 events 1129503 and123602 and Nipponbare (a wild-type control), and the second experimentinvolved progeny lines from T0 events 1164906, 1164914 and 1164922 andNipponbare (a wild-type control.)

Phenotypic Analysis of T1 Lines

Plants height and tiller numbers were measured once every weekpost-transplanting until seed set was achieved. FIGS. 5 a, b, c, d and edepict the growth parameters observed for these plants in two separateexperiments. Transgenic ORF54 plants (T1) do not appear to be toodifferent from the wildtype control (Nipponbare) in terms of plantheight (FIG. 5 a and d). However tillering capacity of ORF54 plants (T1)appear to be higher than in the wild-type control (Nipponbare) (FIG. 5 band e). A closer analysis revealed that 12.5% of the ORF54 T1 plants hadout-performed the best tillering wild-type control plant (FIG. 5 c) withtiller numbers more than doubling. As a result, the biomass as measuredby dry matter production in ORF54 plants (T1) also increased (FIG. 6 a).On an average, the increase in biomass amounts to roughly 66% ascompared to the wild-type control (Nipponbare). Once again 12.5% of theORF54 T1 plant population was seen to produce more dry matter than thehighest dry matter yielding wild-type control (Nipponbare) (FIG. 6 b).In conclusion down-regulation of ORF54 gene expression, or that ofvariants of ORF54, in planta by anti-sense or similar technology leadsto an increase plant biomass.

Plant Transformation—Ryegrass

Perennial ryegrass (Lolium perenne L. cv Impact) was transformedessentially as described in Bajaj et. al. (Plant Cell Reports, 2006, 25:651-659). Embryogenic callus derived from mersitematic regions of thetillers of selected ryegrass lines and Agrobacterium tumefaciens strainEHA101 carrying a modified binary vector (ORF54, FIG. 4) was used fortransformation experiments. Embryogenic calli were immersed withovernight-grown Agrobacterium cultures for 30 minutes with continuousshaking. Calli resistant to hygromycin were selected after sub-culturingthem on co-cultivation medium for 4 weeks. After selection, theresistant calli were sub-cultured on regeneration medium every 2 weeksuntil the plants regenerated. The regenerants that continued to growafter two or three rounds of selection proved to be stabletransformants. Each regenerated plant was then multiplied on maintenancemedium to produce clonal plantlets and subsequently rooted on MS mediumwithout hormones. A rooted plant from each clone was transferred intocontained glasshouse conditions while retaining a clonal counterpart intissue culture as backup. Eighteen independent transgenic lines (1V1,1V3, 1V5, 1V8, 1V10, 1V11, 1V20, 2V2, 2V4, 2V5, 2V7, 2V8, 2V9, 3V5, 3V7,3V10, 3V11, 3V12) and their non-transgenic control plants (T40, T41 andT101, respectively) have been analyzed in a climate-controlledenvironmental laboratory, where they were assessed for biomassproduction under fully water condition.

Screening for Increased Biomass in Growth Chamber

A plant growth system was built using 500 mm long; 90 mm diameterplastic storm-water pipes. The pipes were placed on a mobile tray andsupported at the sides by ropes and metal frame. The tubes were pluggedat the bottom with rockwool and progressively filled with washed mortarsand using water to achieve uniform packing. At the center of the openend of each tube a clump of perennial ryegrass (5 tillers) was planted.Plants from each event were replicate-planted in three tubes. The plantswere arranged at random, one in each of the three replicates, and grownat 70% relative humidity; 16/8 hours day/night cycle and under 650μmol.m⁻².s⁻¹ light intensity. The plants were irrigated daily once inthe morning with 50 mL Hoagland' s solution (Hoagland and Arnon, 1938)and again in the afternoon with 50 mL plain water. The plants wereacclimated initially for twenty days and then the plants were trimmedback to 15 cm height. All plants were allowed to recover from trimmingfor the next fourteen days. Plant tiller numbers were recorded afterseven and 14 days, respectively from the timed day. After fourteen days,plants were trimmed down to 15 cm height. The harvested samples weredried down at 60° C. for three days and then dry weight recorded. Theplants were allowed to grow under fully watered conditions for anotherfourteen days. Again, the plants were trimmed back to 15 cm height andthen the dry weight of the trimmed sample recorded after drying thesamples at 60° C. for three days. The plants were allowed to grow foranother 13 days and the final data on biomass production (dry matter)recorded by harvesting the plants above 15 cm and drying the samples at60° C. for three days.

More than one-fourth of the transgenic events tested produced morebiomass than wild type plants in each of the harvest. When cumulativegrowth was determined over a period of three harvests, one of thetransgenic line, 3V7, produced more than 470% biomass; while in fourother lines, 2V2; 2V7; and 3V10 the biomass increase ranged from over35% to 95% (see FIG. 9).

The above examples illustrate practice of the invention. It will beappreciated by those skilled in the art that numerous variations andmodifications may be made without departing from the spirit and scope ofthe invention.

REFERENCES

-   Adams et al. 1991, Science 252:1651-1656.-   Chen H, Nelson R S, Sherwood J L. (1994) Biotechniques;16 (4):    664-8, 670.-   Chen et al. 2002, Nucleic Acids Res. 31:101-105-   den Dulk-Ras A, Hooykaas P J. (1995) Methods Mol Biol.; 55: 63-72.-   Lee et al. 2003, PNAS 99:12257-12262-   Lee and Lee, 2003 Plant Physiol. 132: 517-529-   Murashige T, Skoog F (1962) Physiol Plant 15: 473-497-   Notredame C., Higgins, D. and Heringa, J. (2000) J. Mol. Biol., 302,    205-217.-   Richmond and Somerville 2000, Current Opinion in Plant Biology.    3:108-116-   Ruan et al. 2004, Trends in Biotechnology 22: 23-30.-   Schmidt-Eisenlohr H, Domke N, Angerer C, Wanner G, Zambryski P C,    Baron C. (1999) J. Bacteriol.; 181 (24): 7485-92.-   Sun et al. 2004, BMC Genomics 5: 1.1-1.4-   Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CABIOS, 10,    19-29.-   Velculescu et al. 1995, Science 270: 484-487

The above examples illustrate practice of the invention. It will beappreciated by those skilled in the art that numerous variations andmodifications may be made without departing from the spirit and scope ofthe invention.

Summary of Sequences FULL-LENGTH SEQ CODING SEQUENCE ID NO: TYPE SPECIESREFERENCE (NUCLEOTIDES) 1 Polypeptide Lolium perenne ORF54 N/A 2Polypeptide Oryza sativa AK098904.1 N/A 3 Polypeptide Oryza sativaAK065041.1 N/A 4 Polypeptide Oryza sativa AK065992.1 N/A 5 PolypeptideOryza sativa AK065747.1 N/A 6 Polypeptide Oryza sativa AK062069.1 N/A 7Polypeptide Oryza sativa XP466543.1 N/A 8 Polypeptide Arabidopsisthaliana BAB01075.1 N/A 9 Polypeptide Arabidopsis thaliana AAL15211.1/N/A AAK59536.1 10 Polynucleotide Lolium perenne ORF54 Start 302; End1904 11 Polynucleotide Oryza sativa AK098904.1 Start 326; End 1928 12Polynucleotide Oryza sativa AK065041.1 Start 326; End 1928 13Polynucleotide Oryza sativa AK065992.1 Start 136; End 1738 14Polynucleotide Oryza sativa AK065747.1 Start 486; End 2161 15Polynucleotide Oryza sativa AK062069.1 Start 2; End 1004 16Polynucleotide Oryza sativa XP466543.1 Start 184; End 1795 17Polynucleotide Arabidopsis thaliana BAB01075.1 Start 1; End 1597 18Polynucleotide Arabidopsis thaliana AAL15211.1/ Start 1; End 1297AAK59536.1 19 Polynucleotide Vector N/A 20 Polypeptide Consensus, allplants N/A 21 Polypeptide Consensus, N/A dicotyledonous plants

What is claimed is:
 1. A method of producing a plant with increasedbiomass the method comprising the step of reducing the expression, in aplant cell or plant, of a polypeptide comprising the sequence of SEQ IDNO: 20, wherein the reducing of expression leads to the increasedbiomass.
 2. The method of claim 1, wherein the expression is reduced bytransforming a plant cell, or plant, with a polynucleotide that iscomplementary to an endogenous gene encoding the polypeptide, such thatexpression of the polynucleotide results in reduced expression of thepolypeptide and leads to the increased biomass.
 3. The method of claim2, wherein the plant cell or plant is transformed with at least one of:a) a polynucleotide including a sequence encoding of a polypeptidecomprising the amino acid sequence of SEQ ID NO:20; b) a polynucleotidecomprising a fragment, of at least 15 nucleotides in length, of thepolynucleotide of a); and c) a polynucleotide comprising a complement,of at least 15 nucleotides in length, of the polynucleotide of a);wherein the expression of the polynucleotide in the plant cell or plantresults in reduced expression of the polypeptide and leads to theincreased biomass.
 4. A plant produced by the method of claim
 1. 5. Aplant produced by the method of claim 1, wherein the expression of anendogenous polypeptide comprising the amino acid sequence of SEQ IDNO:20 is down-regulated in the plant relative to a non-transformedcontrol plant, and wherein the plant has increased biomass relative to anon-transformed control plant.
 6. The plant of claim 5 that has anincreased number of tillers relative to a non-transformed control plant.7. A method for selecting a plant with altered biomass, the methodcomprising testing of a plant for reduced expression of a polynucleotideencoding a polypeptide comprising the amino acid sequence of SEQ IDNO:20, wherein the reduced expression is indicative of increasedbiomass.